CN102924607B - OGP-AcAP5 fusion protein for treating osteoporosis and thrombosis and nucleic acid for coding same - Google Patents
OGP-AcAP5 fusion protein for treating osteoporosis and thrombosis and nucleic acid for coding same Download PDFInfo
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- CN102924607B CN102924607B CN201210477458.7A CN201210477458A CN102924607B CN 102924607 B CN102924607 B CN 102924607B CN 201210477458 A CN201210477458 A CN 201210477458A CN 102924607 B CN102924607 B CN 102924607B
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Abstract
The invention relates to an OGP-AcAP5 fusion protein for treating osteoporosis and thrombosis and a nucleic acid for coding the OGP-AcAP5 fusion protein, belonging to the fusion protein and the nucleic acid for coding the fusion protein, and aims to overcome the defect that no medicament for simutaneously treating osteoporosis and thrombosis is available at present. The OGP-AcAP5 fusion protein for treating osteoporosis and thrombosis has an amino acid sequence shown as SEQ ID NO:2, and the nucleic acid for coding the OGP-AcAP5 fusion protein has an amino acid sequence shown as SEQ ID NO:1. The OGP-AcAP5 fusion protein and the nucleic acid provided by the invention can be used in the pharmaceutical field.
Description
Technical field
The present invention relates to the nucleic acid of a kind of fusion rotein and this fusion rotein of encoding.
Background technology
Senile osteoporosis sickness rate is higher, and there are 200,000,000 sufferers of osteoporosis face in the whole world, and women is more than the male sex.According to the standard of the World Health Organization (WHO), American National health and nutrition survey (NHANES III, 1988～1994 years) result shows, osteoporosis has a strong impact on life of elderly person quality, in 50 years old above crowds, theirs, can there is osteoporotic fracture in life in 1/2 women, 1/5 the male sex, once patient experience osteoporotic fracture for the first time, the danger of secondary fracture obviously strengthens.Chinese Aged occupy first place, the world, and existing patients with osteoporosis 9,000 ten thousand, accounts for 7.1% of total population.Along with the process of social senilization, the sickness rate of osteoporosis is in rising trend, expects the year two thousand fifty will be increased to 2.21 hundred million, and whole world osteoporotic fracture over half will occur in Asia at that time, and the overwhelming majority is in China.There is scholar, to the year of 1995～1996 years U.S.'s osteoporosis, myocardial infarction, palsy and mammary cancer, number occurs and investigate demonstration, annual generation osteoporotic fracture 1,500,000 times, wherein vertebral fracture is 700,000 times, Wrist fracture 200,000 times, Hip Fracture 300,000 times, other fracture 300,000 times.
Thrombosis is a kind of process that relates to the multifactor variation of many h and E factors interact with each other.And have its singularity for its blood coagulation system of the elderly, the elderly's Fibrinogen (FIB) content, tissue plasminogen activator increase and the increase of Type 1 plasminogen activator inhibitor mixture all causes the elderly's hypercoagulative state, so more easily form thrombus; The incidence of thrombus that wherein women after climacteric is found in research is far above the male sex.
For senile osteoporosis and thrombus, become common frdquently encountered disease, and often existed simultaneously.If patient takes multi-medicament and treats osteoporosis and thrombus simultaneously, easily produce drug antagonism reaction; If stagger, will, by considering the transformation period of medicine, also will consider medicine effective concentration on the other hand, and bring inconvenience to patient medicine time on the one hand.
Lack at present a kind of medicine that can effectively treat osteoporosis and thrombus simultaneously.
Summary of the invention
The present invention will solve and there is no at present simultaneously the effectively defect for the treatment of osteoporosis and thrombus medicine, and a kind of OGP-AcAP5 fusion rotein of osteoporosis and thrombus and the nucleic acid of this fusion rotein of encoding of being used for the treatment of providing.
The present invention is used for the treatment of the aminoacid sequence of OGP-AcAP5 fusion rotein of osteoporosis and thrombus as shown in SEQ ID NO:2.
The nucleotide sequence of OGP-AcAP5 fusion rotein that the present invention is above-mentioned is used for the treatment of osteoporosis and thrombus is as shown in SEQ ID NO:1.
The OGP-AcAP5 fusion rotein that the present invention is used for the treatment of osteoporosis and thrombus contains sOGP (OGP) and Ancylostoma caninum anticoagulant hemepeptide (AcAP5), and totally 94 amino acid, are connected with GlyThr between sOGP and Ancylostoma caninum anticoagulant hemepeptide.
The present invention is used for the treatment of the OGP-AcAP5 fusion rotein of osteoporosis and thrombus can treat osteoporosis and thrombus simultaneously, is applicable to suffering from patient's use of these two kinds of diseases simultaneously.The present invention is used for the treatment of the OGP-AcAP5 fusion rotein microbial preparation of osteoporosis and thrombus, does not produce antagonism reaction, uses safety.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment is used for the treatment of the aminoacid sequence of OGP-AcAP5 fusion rotein of osteoporosis and thrombus as shown in SEQ ID NO:2.
Present embodiment is used for the treatment of the nucleotide sequence of OGP-AcAP5 fusion rotein of osteoporosis and thrombus as shown in SEQ ID NO:1, this nucleotide sequence is synthesized by bio-engineering corporation, makes the plasmid vector pUC (OGP/AcAP5) of the nucleic acid that contains fusion rotein.By pUC (OGP/AcAP5) carrier with BamH I and EcoR I double digestion out (endonuclease reaction system is as shown in table 1), and with BamHI and EcoR I double digestion pGEX-6P-1 (endonuclease reaction system is as shown in table 2); Again present embodiment is used for the treatment of osteoporosis and thrombus OGP-AcAP5 fusion rotein DAN fragment (OGP/AcAP5) and through be connected (ligation system is as shown in table 3) of the vector pGEX-6P-1 of double digestion.
Table 1
Table 2
Table 3
The DAN fragment of fusion rotein is placed to be connected with 16 ℃ of vector pGEX-6P-1 and is spent the night, and obtains vector pGEX-OGP/AcAP5; Then vector pGEX-OGP/AcAP5 is transformed in e. coli bl21 (DE3), selects positive recombinant called after e. coli bl21 (DE3-OGP/AcAP5).
On vector pGEX-6P-1, do not contain KpnI restriction enzyme site, the synthetic pGEX-OGP/AcAP5 of present embodiment all can open for Kpn I single endonuclease digestion, illustrates that the OGP-AcAP5 antigen-4 fusion protein gene that present embodiment is used for the treatment of osteoporosis and thrombus successfully imports in plasmid pGEX-6P-1.Then plasmid pGEX-OGP/AcAP5 is imported in e. coli bl21 (DE3), choose positive colony.
Random 37 ℃ of incubated overnight of picking positive colony e. coli bl21 (DE3-OGP/AcAP5) bacterium colony, extract matter DNA, with KpnI, carry out single endonuclease digestion, and with corresponding blank plasmid pGEX-6P-1 in contrast, the plasmid that contains KpnI restriction enzyme site is carried out to gene sequencing.Examining order entrusts biotech firm to carry out, and e. coli bl21 (DE3-OGP/AcAP5) contains DNA shown in SEQ ID NO:1.
E. coli bl21 (DE3-OGP/AcAP5) is placed in to 28 ℃ of LB substratum and cultivates 15h, then adopt GST tag fusion protein method to carry out the separation and purification of albumen, the purity that present embodiment is used for the treatment of the OGP-AcAP5 fusion rotein of osteoporosis and thrombus is 98%, and fusion protein expression is 38%.
The experiment of present embodiment fusion rotein (OGP-AcAP5 fusion rotein) treatment osteoporosis:
Get female sd inbred rats, under aseptic condition, extract bilateral ovaries, negative control group excision bilateral one fritter fat.After 5 days, get 32 of survival healthy rats, be divided at random 4 groups, 8 every group, the oral medicine once of difference
Negative control group (control): the CMC-Na solution that mass concentration is 0.5%, gavage dosage is 5ml/kg;
Model group (model): the OGP-AcAP5 fusion rotein solution that mass concentration is 0.5%, gavage dosage is 5ml/kg; (the OGP-AcAP5 fusion rotein that is used for the treatment of osteoporosis and thrombus of acquisition dissolves with sterilized water)
Positive controls 1: alendronate sodium (Alen) 5mg/kg.
Positive controls 2: the OGP protein solution that mass concentration is 0.5%, gavage dosage is 5ml/kg.
Successive administration three months, respectively gets 8, rat femur head after execution, immerse in 4% glutaraldehyde fixing, with dentistry diamond saw, femoral head sagittal plane is cut, get its a slice, through cleaning, 10% clorox soaks 6h, ultrasonic cleaning 15min, Gradient elution using ethanol, ether soaks, dry, ion sputtering film coating, SX-40 scanning electron microscopic observation, acceleration voltage 20kV.
Observe osteoporosis therapy contrast and experiment, experimental result is in Table 4, and result shows that model group, positive controls 1 and positive controls 2 all have the osteoporotic effect for the treatment of, and the effect of model group is best.
The comparison of table 4 bone trabecula width and surface of bone amass (X ± SD)
| Group | n | Dosage | Bone trabecula width X ± SD | Bone trabecula area X ± SD |
| Negative control group | 8 | 5ml/kg | 51.74±18.6 | 0.5632±0.0659 |
| Positive controls 1 | 8 | 5ml/kg | 114.37±17.9 | 0.6689±0.0492 |
| Positive controls 2 | 8 | 5ml/kg | 119.48±14.2 | 0.6873±0.0485 |
| Model group | 8 | 5ml/kg | 123.56±15.7 | 0.7273±0.0467 |
It is better that present embodiment fusion rotein (OGP-AcAP5) fusion rotein is treated osteoporosis effect by intravenous administration.
The anti-rat suppository experiment of present embodiment fusion rotein (OGP-AcAP5 fusion rotein):
Get 48 of SD rats, be divided at random 6 groups, 8 every group, blank group, control group 1 (positive drug), control group 2 (AcAP5 polypeptide) and the basic, normal, high dosage group of OGP-AcAP5 fusion rotein.Control group 1 is selected heparin sodium injection (dosage is 1650U/kg), control group 2 (dosage is 200 μ g/kg), the dosage of the basic, normal, high dosage group of OGP-AcAP5 fusion rotein (dosage 5 times increase progressively) is respectively 40 μ g/kg, 200 μ g/kg, 1mg/kg, and blank is organized not administration.All, by intravenous administration, after intravenous administration, immediately the carotid artery of separator well is placed in the detection joint of YLS-14B instrument for generating thrombus in small animal, starts thrombus instrument for generating, with constant current galvanic current stimulation (1mA).Record probe is measured the circulation of blood and is conversed blood vessel blockage degree (timed interval of measurement is 4s, is expressed as a percentage data, and all processes continues 5min) by infrared scan.
Observe antithrombotic therapy experimental result, experimental result is in Table 5, and result shows that the carotid artery chocking-up degree of blank group all use obvious difference with other five groups.
The effect of table 5OGP-AcAP5 fusion rotein Chinese People's Anti-Japanese Military and Political College Rat common carotid thrombus
| Group | n | Dosage | Carotid chocking-up degree (%) |
| Blank group | 8 | 0μg/kg | 100.00±0.00 |
| Control group 1 | 8 | 1650U/kg | 50.38±42.19 |
| Control group 2 | 8 | 200μg/kg | 36.43±24.72 |
| Fusion rotein low dose group | 8 | 40μg/kg | 38.69±41.80 |
| Dosage group in fusion rotein | 8 | 200μg/kg | 25.98±41.83 |
| Fusion rotein high dose group | 8 | 1mg/kg | 10.99±11.96 |
Present embodiment fusion rotein (OGP-AcAP5 fusion rotein) toxicity test:
Harbin Medical University's Experimental Animal Center provides SPF level kunming mice.Get mouse that 60 6 week age, male and female half and half, weights are 18 ± 2g as experimental subjects.Adopt maximum tolerance administration (according to clinical Interferon, rabbit consumption 50ug/kg).Experimental group injection total dose 1mg/kg, once daily 0.02mL.Control group mice injection Isodose physiological saline.
Administration process small mouse is acted normally, feed, drinking-water is normal, urine excrement is normal, hair color along white and glossy, activity freely, between reactive good, mouse without mutually baiting phenomenon.Raise 14 days and 30 days continuously, mouse does not all occur dead.
The administration of OGP-AcAP5 fusion rotein is after 14 days, 30 days, put to death mouse, pluck eyeball and get blood, 3000r/min, centrifugal 5min, draw serum, with Beckman automatic clinical chemistry analyzer, detect Main Biochemical: aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), blood urea nitrogen (BUN), uric acid (URCA), total bilirubin (TBIL), TOTAL BILE ACID (TBA).Blood biochemistry index is as shown in table 6.
Table 6
The administration of OGP-AcAP5 fusion rotein is put to death mouse after 14 days, 30 days, cores, liver, spleen, lung, kidney, observes internal organs color, form, calculates each organ coefficient.According to statistics sample, estimate, in every group, randomly draw 7 mouse, core, liver, spleen, lung, kidney HE dyeing do histopathologic examination; Get myeloid tissue Wright's staining observation of cell and have or not oedema, sex change, necrosis etc.The pathological observation result of main organs is as shown in table 7.
Table 7
| Group | Number of days | The heart | Liver | Spleen | Lung | Kidney |
| Control group | 14 | 3.59±0.61 | 32.02±2.39 | 4.47±0.90 | 2.71±0.94 | 7.73±0.95 |
| Experimental group | 14 | 3.48±0.89 | 30.61±3.21 | 4.68±0.73 | 2.94±0.79 | 7.72±0.88 |
| Control group | 30 | 3.62±0.70 | 32.12±2.20 | 4.59±0.88 | 2.92±0.61 | 7.85±0.77 |
| Experimental group | 30 | 3.53±0.90 | 30.66±3.19 | 4.48±0.66 | 2.94±0.58 | 7.88±0.82 |
The HE dyeing of experimental group and the control group main organs heart, liver, spleen, lung, kidney and marrow Wright dyeing paired observation are found: each heart, liver, spleen, lung, kidney and marrow of organizing mouse is all without abnormal changes such as oedema, sex change, necrosis.
This experiment is observed the pathological section of the heart, spleen, lung, kidney and bone marrow smear, is showed no obvious damaging change.The organ coefficient statistical analysis of 5 kinds of internal organs is shown to each group difference is without significance.All illustrate that OGP-AcAP5 fusion rotein and meta-bolites thereof do not produce organic lesion to internal organs.Each group difference of Biochemistry test is without significantly illustrating that OGP-AcAP5 fusion rotein is to liver, the infringement of kidney non-functional.Table OGP-AcAP5 fusion rotein OGP-AcAP5 fusion rotein good biocompatibility, to mouse without obviously acute toxicity, long term toxicity.
At present application of Ancylostoma caninum anticoagulant hemepeptide (anticoagulant peptide, AcAPs) have AcAP5, AcAP6 and tri-kinds of recombinant proteins of AcAPc2.Present embodiment is selected AcAP5 and has been removed its original signal peptide sequence, by 77 amino acid, formed, AcAP5 is the efficient specific inhibitor of Xa factor (thrombin), with Xa react position district at peptide 37-42 (C-R-S-P-G-C-), action site is between the 40th arginine and the 41st glycine.The main anticoagulant factor of AcAP5 Xa, thus be directly combined specificity inhibition Xa with the active centre of Xa, and the common pathway of blocking-up blood coagulation, reaches blood coagulation resisting function.
SOGP (OGP) has the function such as skeletonization and hemopoietic in the external osteoblastic proliferation of promotion, body, is 14 aminoacid small peptides.
Present embodiment fusion rotein inserts GlyThr between sOGP and Ancylostoma caninum anticoagulant hemepeptide, has changed the secondary structure of polypeptide, but does not only make AcAP5 and OGP loss of biological activity, has improved on the contrary its biological activity.The expression amount of present embodiment fusion rotein in intestinal bacteria is also high than single AcAP5 or OGP expression amount.
The effect of present embodiment fusion rotein aspect treatment osteoporosis and thrombus also surpasses single AcAP5 or OGP.Present embodiment fusion rotein can carry out administration by injection or oral mode, does not have first pass effect.
The change of secondary structure does not produce toxicity in vivo, and present embodiment fusion rotein has security; And the change of secondary structure do not affect chromatography and purifying, present embodiment fusion rotein has separation and purification and is easy to feature.
The medicine using in present embodiment, reagent, enzyme, competent cell and plasmid etc. are all bought acquisition, if without particular requirement concentration be product annotation concentration.
Embodiment two: present embodiment is used for the treatment of the nucleotide sequence of OGP-AcAP5 fusion rotein of osteoporosis and thrombus as shown in SEQ ID NO:1.
The nucleotide sequence that present embodiment is used for the treatment of the OGP-AcAP5 fusion rotein of osteoporosis and thrombus is synthesized by bio-engineering corporation.
Present embodiment is used for the treatment of the original signal peptide of having removed AcAP5 gene in the nucleic acid order of OGP-AcAP5 fusion rotein of osteoporosis and thrombus, and between sOGP and Ancylostoma caninum anticoagulant hemepeptide, insert Kpn I restriction enzyme site, both can improve the stability of coded fusion rotein, also change the secondary structure of fusion rotein, its biological property is improved simultaneously.
Present embodiment adds respectively BamHI and EcoR I restriction enzyme site in antigen-4 fusion protein gene both sides, and according to the feature of intestinal bacteria preference codon, has redesigned fusion gene coding base sequence.
Claims (2)
1. the OGP-AcAP5 fusion rotein that is used for the treatment of osteoporosis and thrombus, is characterized in that being used for the treatment of the aminoacid sequence of OGP-AcAP5 fusion rotein of osteoporosis and thrombus as shown in SEQ ID NO:2.
2. coding claim 1 is used for the treatment of the nucleic acid of the OGP-AcAP5 fusion rotein of osteoporosis and thrombus, it is characterized in that the sequence of this nucleic acid is as shown in SEQ ID NO:1.
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Non-Patent Citations (6)
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| cappello m et al.anticoagulant partial [Ancylostoma caninum]，GenBank:AAC47318，77 aa linear.《NCBI genbank》.1996,1-2. * |
| Stanssens P et al.anti-coagulant protein 5 precursor [Ancylostoma caninum],GenBank:AAC47082,100 aa linear.《NCBI genbank》.1996,1. * |
| 余琼等.重组人成骨生长肽治疗去卵巢大鼠骨质疏松的实验观察.《中国地方病学杂志》.2004,第23卷(第4期),318-320. |
| 周凌云等.用原核翻译增强子提高人成骨生长肽基因在大肠杆菌中的表达.《哈尔滨医科大学学报》.2003,第37卷(第5期),379-382. |
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| 重组人成骨生长肽治疗去卵巢大鼠骨质疏松的实验观察;余琼等;《中国地方病学杂志》;20040720;第23卷(第4期);318-320 * |
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